Volume 23, Issue 13, Pages 1163-1172 (July 2013) Specific Kinematics and Motor-Related Neurons for Aversive Chemotaxis in Drosophila  Xiaojing J. Gao, Christopher J. Potter, Daryl M. Gohl, Marion Silies, Alexander Y. Katsov, Thomas R. Clandinin, Liqun Luo  Current Biology  Volume 23, Issue 13, Pages 1163-1172 (July 2013) DOI: 10.1016/j.cub.2013.05.008 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Aversive and Attractive Chemotaxes Enrich Turns but Differ in Their Kinematics (A and B) Trajectories of UAS-shits1/+ flies with 10% acetic acid (A) or 2% vinegar (B) in the bottom right quadrant. Each black dot represents the appearance of one fly in one frame, and data were pooled over all periods, as specified in Figure S3. (C) Definition of the orientation angle θ. Red dot represents the odorant source. Dashed arrow represents the approximated direction of the odorant gradient. Solid arrow represents the direction of the velocity. (D and E) Turn segments (red) flanking the aversive turning points in response to 10% acetic acid (D) or attractive turning points in response to 2% vinegar (E). Arrowhead in (D) represents an exemplary turning point. Gray dots are the same as black dots in Figures 1A and 1B. The dashed lines specify the borders (2.5–7.5 cm in D and 5.5–9.5 cm in E) of areas for data collection in Figures 1H–1K. (F–G′) The relation between the distance from the odorant source and the frequency of aversive (F and G′) or attractive (F′ and G) turns in chemotactic aversion (F and F′) and attraction (G and G′). Compared to the air control, the aversive odorant (acetic acid) condition enriches aversive turning points (F), whereas the attractive odorant (vinegar) condition enriches attractive turning points (G). For each bin, the number of aversive turns against the total number of tracked fly positions between the control and the odorant periods were compared with a chi-square test, and only significantly different bins were indicated. (H–K) The temporal profiles of speed (H and J) and angular speed (I and K) around the aversive (H and I) or attractive (J and K) turning points. Solid traces represent the means of all individual data points over time; dashed traces represent mean ± SEM. The comparisons were made between mean speed or angular speed before turning (–0.4 s to –0.2 s) and at every time point after turning (0.17 s to 0.4 s), with the turning point as time 0. Only significantly different time points were indicated. (Wilcoxon test; n = 387 for aversion, n = 203 for attraction). Throughout the paper: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Current Biology 2013 23, 1163-1172DOI: (10.1016/j.cub.2013.05.008) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Odorant Gradients Modulate Turn Initiation and Direction (A and B) Distribution of velocity orientations in the border area (see Figures 1D and 1E) upon turn initiation in aversion (A; n = 2,954 for the control period, n = 2,276 for the odorant period) or attraction (B; n = 4,642 for the control period, n = 4,238 for the odorant period) in the polar coordinate system. Radial coordinate represents the ratio of the relative frequency during the odorant period and the relative frequency during the control period. Angular coordinate represents θ (defined in Figure 1C; in degrees) upon turn initiation. The velocity orientation upon turn initiation is biased toward the odorant source in aversion (arrow pointing to the right in A) and away from the odorant source in attraction (arrow pointing to the left in B) compared to their control periods, respectively. (C) Distribution of Δθ after turning, for events initiated when the flies were moving toward the odorant source (red dot in Figure 1C) during the control (n = 1,368) and aversive (n = 1,322) odorant period. The arrow pointing to the right indicates that the whole distribution during the aversive period is biased to align the flies against the odorant gradient (>0°). (D) Distribution of Δθ after turning, for events initiated when the flies were moving away from the odorant source (red dot in Figure 1C) during the control (n = 1,887) and attractive (n = 2,081) odorant period. The arrow pointing to the left indicates that the whole distribution during the attractive period is biased to align the flies with the odorant gradient (>0°). All statistical significance in this figure was assessed by Wilcoxon test. The comparisons were made between the entire distributions of θ in the control period and the odorant period in (A) and (B) and the entire distributions of Δθ in the control period and the odorant period in (C) and (D). The genotype is UAS-shits1/+, which also serves as a control for Figure 4. Current Biology 2013 23, 1163-1172DOI: (10.1016/j.cub.2013.05.008) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 441 > shits1 Affects Aversive, but Not Attractive, Chemotaxis (A) Definition of the preference index (PI). Each number represents all positions flies visit in a particular quadrant counted over a defined period of time. For aversion, the PI was calculated 2.5–5 min after the odorant onset, when the index reached a steady state (Figure S3). For attraction, the PI was calculated 1–3 min after the odorant onset to reduce the impact of habituation (Figure S3). (B) Strong aversion to 10% acetic acid in control animals is almost completely abolished in 441 > shits1 flies. (C) 441 > shits1 abolishes aversion to 2% ethyl butyrate. (D) 441 > shits1 abolishes learned aversion. To associate the naturally attractive or neutral odorant (0.1% ethyl butyrate) with a negative valence, we repetitively delivered it to the flies coupled with electric shocks right before testing in the arena. (E) 441 > shits1 does not affect the PI to 2% vinegar. Columns are for mean PIs of multiple independent runs; error bars represent SEM (n ≥ 4). (B), (C), and (E): t test was performed with Holm-Bonferroni post hoc correction; (D): two-way ANOVA tests the significance of the interaction between genotype and conditioning. Current Biology 2013 23, 1163-1172DOI: (10.1016/j.cub.2013.05.008) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 441-GAL4 Defines Neurons for the Completion of Aversive Turns (A and B) The enrichment of aversive turns in response to 10% acetic acid is abolished in 441 > shits1 (A) compared to 441-GAL4 (B) and UAS-shits1/+ (Figure 1F) controls. (C and D) The modulation of turn initiation, as measured by the distribution of θ upon turn initiation in the border area, still persists in 441 > shits1 (C; n = 2,765 for the control period, n = 3,341 for the odorant period) compared to 441-GAL4/+ (D; n = 2,552 for the control period, n = 1,897 for the odorant period) and UAS-shits1/+ (Figure 2A) controls. (E and F) Distribution of Δθ after turning, for events initiated when 441 > shits1 flies (E) were moving toward the odorant source (red dot in Figure 1C) during the control (n = 1,142) and aversive (n = 1,631) odorant periods. The modulation of direction is lost compared to 441-GAL4/+ (F) and UAS-shits1/+ (Figure 2C) controls. (G and H) Angular speed peaks during illumination and then falls below baseline level in 441 > ChR2 flies (G; n = 1,016 before illumination, n = 989 after illumination; n values are the same for speed), but not in control flies (H; n = 890 before illumination, n = 852 after illumination; n values are the same for speed). (I and J) Speed increases after illumination in 441 > ChR2 flies (I), but not in control flies (J). Gray bar represents the light-on period. (A) and (B) show the same comparison as in the corresponding panels in Figures 1F and 1G. (C)–(F) show the same comparison as in the corresponding panels in Figures 2A–2D. (G)–(J) show Wilcoxon tests between the average angular speeds (G and H) or speeds (I and J) before (–0.2 s to –0.1 s) and after (0.4 s) illumination, with the light onset being time 0. Current Biology 2013 23, 1163-1172DOI: (10.1016/j.cub.2013.05.008) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 441-GAL4 Targets Redundant Neurons Necessary for the Execution of Aversion (A and A′) 441-GAL4 expression is visualized with a membrane-tagged GFP in the brain (A) and the VNC (A′). Arrowhead shows the ellipsoid body. (B and B′) tsh-GAL80 suppresses VNC expression of 441-GAL4. (C) Aversion in 441 > shits1 flies is partially restored with tsh-GAL80 or EB-GAL80. The first column represents the same data as the last column in Figure 3B. Columns are mean PIs, and error bars represent SEM (n ≥ 4); all tested pairs are labeled; t test was performed with Holm-Bonferroni post hoc correction. (D and D′) R13C06-GAL80 suppresses EB expression of 441-GAL4, as indicated by the open arrowhead. (E1–F2′) Average 441-GAL4 expression without (E1–E2′) or with (F1–F2′) tsh-GAL80 is visualized with a dendritic marker in the ventral (E1, E2, F1, and F2) and dorsal (E1′, E2′, F1′, and F2′) halves of the VNC after image registration against a standard VNC, shown with neuropil counterstaining of VNC (E1, E1′, F1, and F1′) or without (E2, E2′, F2, and F2′). Dashed boxes show abdominal ganglion; arrows show ventromedial cell bodies (E1 and E2; suppression indicated by dashed arrows in F1 and F2) and the corresponding dorsal-bilateral projections (E1′ and E2′; suppression indicated by dashed arrows in F1′ and F2′); arrowhead shows projection along the midline (E1′ and E2′; suppression indicated by open arrowhead in F1′ and F2′). Scale bars represent 50 μm. Current Biology 2013 23, 1163-1172DOI: (10.1016/j.cub.2013.05.008) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 6 Analyses of 918-GAL4 Support the Role of Specific 441+tsh+ Neurons in Aversion (A) 918 > shits1 attenuates aversion to 10% acetic acid, and this attenuation is suppressed by tsh-GAL80 or 441-GAL80. (B) 918 > shits1 abolishes aversion to 2% ethyl butyrate. (C) 918 > shits1 does not affect attraction to 2% vinegar. Columns in (A–C) are mean PIs of multiple independent runs, and error bars represent SEM (n ≥ 3); t test was performed with Holm-Bonferroni post hoc correction. (D) 918-GAL4 expression in the VNC is visualized with a membrane-tagged GFP. Arrows point to the ventromedial cell bodies similar to those in Figures 5E1–5E1′. (E) tsh-GAL80 suppresses VNC expression of 918-GAL4, including the ventromedial class, as indicated by the dashed arrows. (F) 918-GAL4 expression is visualized with a destabilized GFP. Arrows point to the ventromedial cell bodies. (G) 441-GAL80 suppressed the expression of 918-GAL4 in the ventromedial class, as indicated by the dashed arrows. Scale bars represent 50 μm. (H) A schematic summary of the functionality of neuronal populations underlying aversive chemotaxis inferred from our genetic analysis. Each circle and the line attached to it represent a neuronal population and the direction of information flow; dashed lines represent potential intermediate layers of relay neurons. The “OR” logic gate near the end of the aversive circuit reflects the redundant roles of the pathways mediated by EB and dTdc2+ VNC neurons. Current Biology 2013 23, 1163-1172DOI: (10.1016/j.cub.2013.05.008) Copyright © 2013 Elsevier Ltd Terms and Conditions